Triglycerides represent the major form of energy stored in eukaryotes. Disorders or imbalances in triglyceride metabolism are implicated in the pathogenesis of and increased risk for obesity, insulin resistance syndrome and type II diabetes, nonalcoholic fatty liver disease and coronary heart disease (see, Lewis, et al, Endocrine Reviews (2002) 23:201 and Malloy and Kane, Adv Intern Med (2001) 47:111). Additionally, hypertriglyceridemia is often an adverse consequence of cancer therapy (see, Bast, et al. Cancer Medicine, 5th Ed., (2000) B. C. Decker, Hamilton, Ontario, Calif.).
A key enzyme in the synthesis of triglycerides is acyl CoA:diacylglycerol acyltransferase, or DGAT. DGAT is a microsomal enzyme that is widely expressed in mammalian tissues and that catalyzes the joining of 1,2-diacylglycerol and fatty acyl CoA to form triglycerides at the endoplasmic reticulum (reviewed in Chen and Farese, Trends Cardiovasc Med (2000) 10:188 and Farese, et al, Curr Opin Lipidol (2000) 11:229). It was originally thought that DGAT uniquely controlled the catalysis of the final step of acylation of diacylglycerol to triglyceride in the two major pathways for triglyceride synthesis, the glycerol phosphate and monoacylglycerol pathways. Because triglycerides are considered essential for survival, and their synthesis was thought to occur through a single mechanism, inhibition of triglyceride synthesis through inhibiting the activity of DGAT has been largely unexplored.
Genes encoding mouse DGAT1 and the related human homologs ARGP1 and ARGP2 now have been cloned and characterized (Cases, et al, Proc Natl Acad Sci (1998) 95:13018; Oelkers, et al, J Biol Chem (1998) 273:26765). The gene for mouse DGAT1 has been used to create DGAT knock-out mice to better elucidate the function of the DGAT gene. Unexpectedly, mice unable to express a functional DGAT enzyme (Dgat−/− mice) are viable and still able to synthesize triglycerides, indicating that multiple catalytic mechanisms contribute to triglyceride synthesis (Smith, et al, Nature Genetics (2000) 25:87). Other enzymes that catalyze triglyceride synthesis, for example, DGAT2 and diacylglycerol transacylase, also have been identified (Buhman, J Biol Chem, supra and Cases, et al, J Biol Chem (2001) 276:38870).
Significantly, Dgat−/− mice are resistant to diet-induced obesity and remain lean. Even when fed a high fat diet (21% fat) Dgat−/− mice maintain weights comparable to mice fed a regular diet (4% fat) and have lower total body triglyceride levels. The obesity resistance in Dgat−/− mice is not due to deceased caloric intake, but the result of increased energy expenditure and decreased resistance to insulin and leptin (Smith, et al, Nature Genetics, supra; Chen and Farese, Trends Cardiovasc Med, supra; and Chen, et al, J Clin Invest (2002) 109:1049). Additionally, Dgat−/− mice have reduced rates of triglyceride absorption (Buhman, et al, J Biol Chem (2002) 277:25474). In addition to improved triglyceride metabolism, Dgat−/− mice also have improved glucose metabolism, with lower glucose and insulin levels following a glucose load, in comparison to wild-type mice (Chen and Farese, Trends Cardiovasc Med, supra).
The finding that multiple enzymes contribute to catalyzing the synthesis of triglyceride from diacylglycerol is significant, because it presents the opportunity to modulate one catalytic mechanism of this biochemical reaction to achieve therapeutic results in an individual with minimal adverse side effects. Compounds that inhibit the conversion of diacylglycerol to triglyceride, for instance by specifically inhibiting the activity of the human homolog of DGAT1, will find use in lowering corporeal concentrations and absorption of triglycerides to therapeutically counteract the pathogenic effects caused by abnormal metabolism of triglycerides in obesity, insulin resistance syndrome and overt type II diabetes, congestive heart failure and atherosclerosis, and as a consequence of cancer therapy.
Because of the ever increasing prevalence of obesity, type II diabetes, heart disease and cancer in societies throughout the world, there is a pressing need in developing new therapies to effectively treat and prevent these diseases. Therefore there is an interest in developing compounds that can potently and specifically modulate a single catalytic mechanism of the enzymatic conversion of diacylglycerol to triglyceride. Of particular promise are compounds that specifically inhibit the catalytic activity of DGAT1 and its other mammalian homologs.